Cell surface receptors coupled to the heterotrimeric GTP-binding proteins are universally responsible for the transmembrane transmission of extracellular messengers such as hormones, neurotransmitters and sensory stimuli. Because of this direct involvement in the regulation of the most crucial cellular functions, G-protein coupled receptors (GPCRs) are among the most important targets of therapeutic intervention. It's estimated that about 50% of drugs in use act on GPCRs. Thus, understanding of the receptor and the G-protein functions at the molecular level is among the highest priorities of public health research. Several competing models aim at describing the universal mechanism of G-protein activation by GPCRs, but none has presented compelling and conclusive experimental evidence so far. HYPOTHESIS: G-protein beta-gamma subunit complex is a key molecular switch at the center of the gear-shift model of G-protein activation. We will test this hypothesis using the prototypical GPCR rhodopsin (R) and the G-protein transducin (Gt) responsible for phototransduction in retinal rod cells as a model system. Four interconnected Specific Aims will test various aspects of the hypothesis, such as questions of the molecular organization of the receptor-G-protein complex, the high-resolution picture of the receptor-G-protein interface, the mechanism of signal transfer from the receptor, and the roles of individual G-protein subunits, especially the G-protein beta-gamma subunit complex, in this dynamic process.